Although it was previously demonstrated that T cell receptor-mediated interaction is necessary to thymic stromal development, the role of costimulatory signals in thymic development has not been assessed. Studies were therefore undertaken to determine whether costimulation influences cortico-medullary organogenesis and thymic repertoire selection. Mice with genetically altered expression of costimulatory molecules CD80 (B7-1), CD86 (B7-2), and CD40, or of the costimulatory receptors CD28 and CD154 (CD40 ligand) have been analyzed for thymic development and expressed T cell repertoire. Disruption of the CD28-B7 pathway or of the CD40-CD154 pathway had minimal effect on development of normal thymic cortico-medullary architecture. In contrast, disruption of both pathways resulted in a profound failure of thymic development. These findings identify a previously unappreciated role of redundant costimulatory pathways mediating an essential function in thymic development. The effect of these costimulatory pathways on selection of the T cell repertoire was studied in parallel. Two pathways of negative selection have been identified. One pathway is CD40L-dependent and acts at a relatively early stage in intra-thymic development and is mediated by a non-cell-autonomous mechanism. The second is CD40L-independent and occurs later in intra-thymic or post-thymic development. Interestingly, we have observed that when negative selection is abrogated by inactivation of the CD40L pathway, functional self tolerance is maintained by a non-deletional mechanism. This non-deletional tolerance is mediated by a CD28-dependent pathway. Thus, redundant pathways exist to protect against self reactivity in the thymus, and disruption of both C40L (deletional) and CD28 (non-deletional) mechanisms of self tolerance results in a population of highly self-reactive thymic T cells. These findings have elucidated the roles of costimulatory pathways in thymic T cell repertoire selection and self tolerance. Most recently, we have analyzed the signals that mediate T cell-epithelial cell cross-talk during thymic development. We have observed that CD28 costimulation is essential for optimal induction of TNFab in single-positive (SP) thymocytes, and that combined defects in CD40-CD40L and TNFab pathways result in defects in medullary thymic eipthelial cell (mTEC) development that are similar to those in combined CD40-CD40L and CD28-B7 disruption, and are in fact as profound as those seen in complete absence of mature SP TCRab thymocytes. We found that selective inactivation in TEC of Traf3, an inhibitor of alternative NFkB signaling, restores mTEC development in the complete absence of SP thymocytes and does so through a RelB-dependent pathway. These findings indicate a novel role for CD28-B7 in addition to other mediators of T cell-TEC cross-talk and demonstrate that any and all signals provided by matue thymocytes for mTEC development are provided by activation of RelB-dependent alternative NFkB signlaing. The expression of both CD28 and B7 is highly regulated during thymic development, such that the most immature thymocytes are protected from exposure to costimulatory signaling. The ataxia telangiectasia mutated (ATM) protein plays a central role in sensing and responding to chromatin changes including DNA double strand breaks (dsb) generated by insults such as ionizing radiation. Lymphoid cells are unique in undergoing physiologic dsb in the processes of Ig class switch recombination (CSR) and T or B cell receptor V(D)J recombination, and a role for ATM in these processes has been suggested by clinical observations in ataxia telangiectasia (AT) patients with mutations in the Atm gene as well as by studies of mice deficient in ATM. We have characterize a defect in T cell development in ATM-deficient mice that is associated with decreased efficiency in V-J rearrangement of the endogenous T cell receptor (TCR)-alpha locus, with consequent reduction in the number of mature TCR-expressing CD4+ and CD8+ thymic T cells. CD4+CD8+ thymocytes from ATM-deficient mice exhibit a reduction in TCRalpha V-J rearrangement as measured by real-time genomic PCR, while containing an increased frequency of unresolved TCR Jbeta coding ends (CE), indicating a delay in TCRalpha V-J coding joint formation. The absence of ATM thus results in reduced frequency of successfully completed TCRalpha recombination, leading to decreased TCRalpha/beta expression and decreased positive selection of mature CD4+ and CD8+ T cells, defining a pivotal role for ATM in T cell development. We have subsequently analyzed the role of ATM in earlier stages of T cell development, including TCR beta rearrangement. In the absence of ATM, VDJ rearrangement of TCRb is defective during the early DN2/3 stage of thymocyte development. This defect has been characterized by direct immuno-FISH visualization of increased DSB foci at the TCRb locus, by decreased productive VDJ recombination, and by decreased expression of TCRb protein. This defect in VDJ recombination reults in functional outcomes that include defective DN3-DN4 and DN-DP developmental transitions, as well as in altered TCRb repertoire as analyzed by deep sequencing of TCRb CD3 regions. These findings thus provide novel insights into the mechanism and functional consequences of ATM during T cell development. Most recently, we have characterized the expression of novel hybrid TCR chains generated by trans-recombination of two distinct TCR loci. Trans recombined genes are expressed at the level of cell surface receptors, undergo MHC-restricted thymic selection, and support naive and memory mature T cell development. Transgenic mice expressing hybrid receptors are being generated to further define the function of these novel receptors. Costimulatory pathways play critical roles during lymphocyte development as well as during differentiaion of mature immune responses. The dissection of specific cell interaction pathways mediating these effects is, however, incomplete. To dissect in detail the nature of these interactions, we have initiated the generation of conditional knock-out and mutant transgenic mouse models, which have not previously existed. To date, we have generated the first (to our knowledge) conditional CD80 (B7-1) knockout mice and have begun the charactrization of cell-specific B7 expression during T cell development, and in functional properties of T cells, B cells, dendritic cells, andthymic epithelium. Early results have identified previously undescribed distinct pathways for T helper-dependent antibody responses as elucidated by their costimulatory requirements. We have very recently used a similar strategy to generate conditional CD40 knockouts, with founders now being analyzed.

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National Cancer Institute Division of Basic Sciences
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